Proteins and lipids are the major constituents of egg yolk. Both fractions play important roles in the food processing, cosmetic, and pharmaceutical industry. The most plentiful constituent in egg yolk solids is the lipid fraction which constitutes approximately 1/3 of the raw yolk. The lipid is comprised mainly of various phospholipids containing mainly phosphatidylcholine or lecithin, then by cholesterol, a relatively minor constituent in the egg yolk. Proteins constitute 15% to 17% of the yolk, including phosvitin, .alpha.- and .beta.-lipovitellins and low density lipoproteins in the yolk granule and .alpha.-.beta.-, and .gamma.-livetins as well as lipoproteins in the yolk plasma. .gamma.-livetin has been recognized as the IgG-like antibody and is also commonly referred to a yolk immunoglobulin or IgY.
Due to the ready availability of egg yolk, many attempts have been made to isolate phospholipids (lecithin), IgY, and other bioactive or functional compounds. However, it is difficult to recover lipid components without denaturing or inactivating the egg yolk protein components.
Several methods for the extraction of phospholipids from egg yolk are known to those skilled in the art: the scorching method; solvent extraction of liquid egg yolk; and solvent extraction of dry egg yolk.
The scorching method comprises direct heating of egg yolk, either liquid or cooked, until the egg yolk proteins scorch. The yolk proteins are of no further value after this process. The extraction efficiency of this process is low because a large amount of egg lipids remain adhered to the protein residue. Furthermore, the extraction process results in deterioration of the unsaturated fatty acids because of the high temperature and exposure to the atmosphere. For these reasons, the scorching process has not been implemented on a commercial basis to extract egg phospholipids.
Solvent extraction of liquid egg yolk using a mixture of polar and non-polar solvents has been effectively applied to the extraction of egg lipids, which contain the desired egg yolk lecithin. Since the extraction process uses a mixture of solvents, the procedures for recovery are complicated and cannot be conducted economically. Further, solvents used may have boiling points sufficiently high to expose the egg lipid to temperatures in which oxidation of the valuable components occurs readily. During the heating process to remove the solvents, the egg yolk extract typically changes in color from yellow to orange to brown. The quality of the egg yolk lecithin obtained through this process may be extremely poor.
In the past the industry has practiced solvent extraction of lecithin from a yolk, by the use of dried yolk as a starting material, where the dried yolk was obtained by drying a raw yolk with heated air. The dried yolk was subjected to extraction with a solvent mixture of a polar solvent such as methanol or acetone in a non-polar solvent such as chloroform, diethyl ether or trichlorolethylene. Since this solvent mixture contains two different kinds of component solvents, the procedures for recovery of the component solvents during the extraction process are complicated and cannot be conducted economically. Further, solvents used may have boiling points in excess of 35.degree. C. Therefore, for complete removal of the solvents from the extract, the extract may be required to be heated to high temperatures through use of steam or to be subjected to exposure of high temperatures for prolonged periods of time under reduced pressure. Yolk lecithin is extremely susceptible to oxidation. The yolk lecithin may be required to be heat-treated at a temperature as high as 60.degree. C. or more under atmospheric pressure to remove residual solvent. During this heat treatment color of the yolk lecithin generally changes from yellow to orange to brown. The quality of the yolk lecithin obtained through the heat treatment for the removal of the solvents may be extremely poor, the yolk lecithin produced by such conventional solvent extraction methods also may smell bad and be colored brown. The conventional process for lecithin and lipid extraction utilizes organic solvents, whereby proteins are inevitably denatured. Typically, egg yolk is dried, usually by spray drying. The powder is extracted with a short chain alcohol, such as methanol or ethanol, or other suitable solvent(s) to yield a crude egg oil. This oil contains the desired phospholipids. The extraction process results in non-functional, denatured egg protein that can not be used for typical functional applications.
An alternative to conventional solvent extraction procedures for separating lecithin and cholesterol from egg oil is the use of supercritical fluid extraction, however, even under these conditions, some proteins are denatured.
Since egg yolk is a mixture of lipids, lipid proteins and water-soluble proteins, the water-soluble fraction including IgY may be extracted in undenatured form with water before further fractionation of the water soluble components. These procedures use a water dilution process with pH and salt adjustments. These processes are optimized to separate the IgY into the supernatant portion while directing the lipids, including phospholipids, into the pellet fraction. These processes typically utilize an acidic pH. The phospholipids may be further recovered from the pellet by resuspension and additional extraction. This additional work to recover the phospholipids is not practical on an industrial scale. Some attempts have also been made to separate the .gamma.-livetins from egg yolk by using polyethylene glycol or sodium dextran sulfate.
Egg yolk lipids are suitable for food, cosmetic, and pharmeceutical application. Each of these applications may require a different composition of the lecithin fraction. In order to obtain these variations, it is necessary to selectively obtain egg which contain the desired egg yolk lecithin. One type application which may require a specific egg yolk lecithin composition is for the use in infant formula. Egg lipids may be a logical source of lipid to enable a product to be made that is analogous in fatty acid composition to the lipid constituents of human milk. Human milk contains a variety of chain elongated--unsaturated fatty acids derived from C.sub.18:2.omega.6 and C.sub.18:30.omega.3. These polyunsaturated homologs of the essential fatty acids are not found in presently available commercial formulas. A need therefore exists to prepare an oil which could provide these constituents for use in infant formula as derived from egg oil.
In general the yolk of various strains of chicken eggs is 30% to 36% lipid comprising about 65% triglycerides, 28.3% phospholipids and 5.2% cholesterol. The total amount of saturated fatty acid is approximately 40% of the fatty acids. Major unsaturated fatty acids are oleic, linoleic, and linolenic with a small quantity of C.sub.20.omega.6, C.sub.22.omega.6 and C.sub.22.omega.3 polyunsaturated fatty acids. In recent years, efforts have been directed to produce a milk-based infant formula which is similar to human milk. In general, a dietary product is required which may be added to commercially available cow's milk. The dietary product is intended to adapt the cow's milk to the requirements of human nursing and to make the cow's milk similar to human milk. A lipid component of the dietary product is preferably a mixture of vegetable oil with soy lecithin and eventually with milk fats.
A problem with the past is that the oils used as the additive do not contain C.sub.20 and C.sub.22 omega-6 and omega-3 fatty acids which are present in human milk. Accordingly, these dietary preparations do not provide a balance of fatty acids that are similar in composition to that of human milk.
Studies have revealed that an adequate intake of mother's own milk could produce from 90 milligrams to 130 milligrams of C.sub.20 and C.sub.22 omega-6 fatty acids per day and 55-75 milligrams of C.sub.20 and C.sub.22 omega3 fatty acids per day. In view of these studies, dietary formulas for the premature/and pre-term infant should contain C.sub.20 and C.sub.22 omega-6 fatty acids and C.sub.20 and C.sub.22 omega-3 fatty acids to promote healthy growth. Fatty acids may have particular significance for the synthesis of structural lipids in brain tissue, however, these studies do not suggest any means for determining the quantities of these fatty acids required in infant formula. It may be advisable to provide essential fatty acids in a dietary formula for premature infants to insure normal synthesis of nerve tissues. It is particularly important that the quantifies of C.sub.20 and C.sub.22 omega-6 and omega-3 fatty acids be limited to appropriate ranges in the dietary formula, therefore, over feeding of these fatty acids should be avoided to minimize complications with prostaglandin metabolism and other adverse effects on an infant's physiology.
Omega-3 highly unsaturated fatty acids are of significant commercial interest in that they had been recently recognized as important dietary compounds for preventing arterialsclerosis and coronary heart disease, for alleviating inflammatory conditions and for retarding the growth of tumor cells. These beneficial effects are a result of both the omega-3 highly unsaturated fatty acids causing competitive inhibition of compounds produced from omega-6 fatty acids and from beneficial compounds produced directly from the omega-3 highly unsaturated fatty acids themselves. Omega-6 fatty acids are the predominant highly unsaturated fatty acids found in plants and animals. Currently the only commercially available dietary source of omega-3 highly unsaturated fatty acids is from certain fish oils which can contain up to 20% to 30% of these fatty acids. Beneficial effects of these fatty acids may be obtained by eating fish several times during a week or by daily intake of concentrated fish oil. Consequently, large quantities of fish oil are processed and capsulated each year for sale as a dietary supplement.
Several significant problems exist with these fish oil supplements. First, fish oil supplements may contain high levels of fat-soluble vitamins which are found naturally in fish oils. When ingested, these vitamins are stored and metabolized as fat in the human body rather than excreted. High doses of these vitamins may be unsafe, leading to kidney problems or blindness. Secondly, fish oils containing up to 80% of the saturated and omega-6 fatty acids both of which may have deleterious health affects. Additionally, fish oils have a strong fishy taste and odor and as such cannot be added to processed foods as a food additive without negatively effecting the taste of the food product. Moreover, the isolation of pure omega-3 highly unsaturated fatty acids is a relatively expensive process resulting in very high prices.
The present invention is directed to a method for separation of an egg yolk into desired fractions. The egg yolk contains many beneficial molecules which are valued for functional and biological characteristics following consumption. An unprocessed and uncooked egg yolk, in general, comprises water, yolk proteins and egg lipids. The egg yolk lipids comprises neutral lipids, yolk lecithin, sterols, and cholesterol. The conventionally proposed methods for removal of egg lipids from raw egg yolk are classified into two kinds of methods, namely the scorching method and a solvent extraction method.
In the past, the separation of an egg yolk into desired fractions, where each fraction had different functional and practical purposes, occurred through the method of utilizing solvents, or conditions which in turn caused the protein fractions of the egg yolk to denature. A method has not been previously known for separation of an egg yolk into desired fractions through processes utilizing mild food grade solvents which did not cause the resulting protein fractions to denature.
In the past, egg yolk phospholipids were separated from egg yolk by a tedious extraction process. In this extraction process, the egg yolk was first dried. The dried powder was then solvent extracted with a polar solvent such as ethanol, to remove the phospholipids. The solvent was then evaporated to yield phospholipids while the remainder of the egg yolk powder was no longer suitable as a functional food ingredient.